Circadian rhythms provide organisms with an adaptive advantage, enhancing the health and fitness of individuals by ensuring that diverse physiological processes occur at the most appropriate times of day. The long-term objective of this proposal is to understand the molecular basis of circadian rhythms in eukaryotic cells. We have identified key regulators of the circadian clock in our previous studies, and will now use genomic, biochemical, genetic, and mathematical modeling approaches to appropriately place these proteins in the circadian system. We will conduct our studies in Arabidopsis thaliana, a model plant that is uniquely well-suited for these experiments due to its compact genome, extensive genetic and genomic resources, and ability to tolerate mutations in chromatin regulatory pathways that are lethal to other complex eukaryotes. We will first use genomic, biochemical, and genetic approaches to characterize the role of a protein conserved across eukaryotes (but of unknown biochemical function) in the regulation of chromatin. We anticipate this work will generate insights into mechanisms governing chromatin organization and thus gene expression in diverse eukaryotes. Next, we will use genetic and biochemical techniques to investigate the roles of a family of related transcription factors in the workings of the circadian oscillator. In the process, we will test predictions made by a mathematical model, evaluating how well this model describes the regulatory relationships that drive the clock. Finally, we will modify an existing competitive chromatin immunoprecipitation protocol to investigate how the binding dynamics of antagonistic transcription factors to chromatin shape the dynamic regulation of gene expression in vivo. These experiments will reveal general principles governing the temporal regulation of gene expression and thus will be applicable to many organisms and processes. Our exploration of shared mechanisms that control circadian clock function in diverse organisms will increase our understanding of how clocks function in humans and shed light on how they promote human health and welfare.